Oligomerization Is Crucial for the Stability and Function of Heme Oxygenase-1 in the Endoplasmic Reticulum

Heme oxygenase-1 (HO-1), a stress-inducible enzyme anchored in the endoplasmic reticulum (ER) by a single transmembrane segment (TMS) located at the C terminus, interacts with NADPH cytochrome P450 reductase and biliverdin reductase to catalyze heme degradation to biliverdin and its metabolite, bilirubin. Previous studies suggested that HO-1 functions as a monomer. Using chemical cross-linking, co-immunoprecipitation, and fluorescence resonance energy transfer (FRET) experiments, here we showed that HO-1 forms dimers/oligomers in the ER. However, oligomerization was not observed with a truncated HO-1 lacking the C-terminal TMS (amino acids 266–285), which exhibited cytosolic and nuclear localization, indicating that the TMS is essential for the self-assembly of HO-1 in the ER. To identify the interface involved in the TMS-TMS interaction, residue Trp-270, predicted by molecular modeling as a potential interfacial residue of TMS α-helices, was mutated, and the effects on protein subcellular localization and activity assessed. The results showed that the W270A mutant was present exclusively in the ER and formed oligomers with similar activity to those of the wild type HO-1. Interestingly, the W270N mutant was localized not only in the ER, but also in the cytosol and nucleus, suggesting it is susceptible to proteolytic cleavage. Moreover, the microsomal HO activity of the W270N mutant was significantly lower than that of the wild type. The W270N mutation appears to interfere with the oligomeric state, as revealed by a lower FRET efficiency. Collectively, these data suggest that oligomerization, driven by TMS-TMS interactions, is crucial for the stabilization and function of HO-1 in the ER.Heme oxygenase (HO)³ catalyzes the NADPH cytochrome P450 reductase-dependent oxidative degradation of cellular heme to biliverdin, carbon monoxide (CO), and free iron (1, 2). Biliverdin is subsequently converted to bilirubin by biliverdin reductase in the cytosol. Two HO isoforms have been identified in mammalian systems. HO-1 is a 288 amino acid protein and is expressed at high amounts in a variety of pathological conditions associated with cellular stress. There is compelling evidence that HO-1 induction represents an important cytoprotective defense mechanism against oxidative insults by virtue of the anti-oxidant properties of the bilirubin and the anti-inflammatory effect of the CO produced (2). HO-1 is anchored in the endoplasmic reticulum (ER) through a single transmembrane segment (TMS) located at the C terminus, while the rest of the molecule is cytoplasmic (3). HO-1 is sensitive to proteolytic cleavage (4), and it was recently shown that HO-1 can be proteolytically cleaved from the ER and translocated to the nucleus under certain stress conditions (5). Although the catalytic site in the cytoplasmic domain remains intact, the activity of soluble HO-1 is drastically reduced (5), indicating that ER localization is important for its full enzymatic function.Self-assembly to form dimers and higher oligomers is a common phenomenon in many membrane proteins (6, 7). Numerous studies have revealed that interactions between TMSs play an important role in the structure and function of many membrane proteins. Examples include receptors, enzymes, neurotransmitter transporters, and ion channels, in which oligomerization is crucial for their proper cellular localization and function (8). HO-1 does not contain any cysteine residues and has therefore been assumed to function as a monomer (1). To determine whether HO-1 forms oligomers in native membranes, in the present study, we performed chemical cross-linking, co-immunoprecipitation, and FRET analysis using fluorescent protein tags fused to the N terminus of HO-1. The results showed that HO-1 formed dimers/oligomers in the ER and that the TMS provided the interface for the protein-protein interactions. Interference with the TMS-TMS interaction resulted in destabilization of HO-1 and a reduction in enzymatic function.     

Profiling gene expression induced by protease-activated receptor 2 (PAR2) activation in human kidney cells

Protease-Activated Receptor-2 (PAR2) has been implicated through genetic knockout mice with cytokine regulation and arthritis development. Many studies have associated PAR2 with inflammatory conditions (arthritis, airways inflammation, IBD) and key events in tumor progression (angiogenesis, metastasis), but they have relied heavily on the use of single agonists to identify physiological roles for PAR2. However such probes are now known not to be highly selective for PAR2, and thus precisely what PAR2 does and what mechanisms of downstream regulation are truly affected remain obscure. Effects of PAR2 activation on gene expression in Human Embryonic Kidney cells (HEK293), a commonly studied cell line in PAR2 research, were investigated here by comparing 19,000 human genes for intersecting up- or down-regulation by both trypsin (an endogenous protease that activates PAR2) and a PAR2 activating hexapeptide (2f-LIGRLO-NH(2)). Among 2,500 human genes regulated similarly by both agonists, there were clear associations between PAR2 activation and cellular metabolism (1,000 genes), the cell cycle, the MAPK pathway, HDAC and sirtuin enzymes, inflammatory cytokines, and anti-complement function. PAR-2 activation up-regulated four genes more than 5 fold (DUSP6, WWOX, AREG, SERPINB2) and down-regulated another six genes more than 3 fold (TXNIP, RARG, ITGB4, CTSD, MSC and TM4SF15). Both PAR2 and PAR1 activation resulted in up-regulated expression of several genes (CD44, FOSL1, TNFRSF12A, RAB3A, COPEB, CORO1C, THBS1, SDC4) known to be important in cancer. This is the first widespread profiling of specific activation of PAR2 and provides a valuable platform for better understanding key mechanistic roles of PAR2 in human physiology. Results clearly support the development of both antagonists and agonists of human PAR2 as potential disease modifying therapeutic agents.     

Fibrobacter communities in the gastrointestinal tracts of diverse hindgut‐fermenting herbivores are distinct from those of the rumen

The genus Fibrobacter contains cellulolytic bacteria originally isolated from the rumen. Culture‐independent investigations have since identified Fibrobacter populations in the gastrointestinal tracts of numerous hindgut‐fermenting herbivores, but their physiology is poorly characterized due to few representative axenic cultures. To test the hypothesis that novel Fibrobacter diversity exists in hindgut fermenters, we performed culturing and 16S rRNA gene amplicon sequencing on samples collected from phylogenetically diverse herbivorous hosts. Using a unique approach for recovering axenic Fibrobacter cultures, we isolated 45 novel strains from 11 different hosts. Full‐length 16S rRNA gene sequencing of these isolates identified nine discrete phylotypes (cutoff = 0.03%) among them, including several that were only isolated from hindgut‐fermenting hosts, and four previously unrepresented by axenic cultures. Our phylogenetic analysis indicated that six of the phylotypes are more closely related to previously described subspecies of Fibrobacter succinogenes, while the remaining three were more closely related to F. intestinalis. Culture‐independent bacterial community profiling confirmed that most isolates were representative of numerically dominant phylotypes in their respective samples and strengthened the association of certain phylotypes with either ruminants or hindgut‐fermenters. Despite considerable phylogenetic diversity observed among the Fibrobacter strains isolated here, phenotypic characterization suggests a conserved specialization for growth on cellulose.     

Soluble enzyme system for vitamin K-dependent carboxylation.

The vitamin K-dependent carboxylating system has been solubilized by Lubrol PX or Triton X-100 treatment of vitamin K-deficient rat liver microsomes. As obtained from vitamin K-deficient rat liver, this soluble preparation is dependent upon the in vitro addition of vitamin K1 for carboxylating activity. The enzyme system is complex and is dependent upon NADH and dithiothreitol for maximum activity. While detergents used to solubilize the enzyme complex do markedly inhibit the activity of the system, the solubilized system is still highly responsive to vitamin K addition and can be used for further study of the carboxylating enzyme system. The requirement for dithiothreitol and the inhibition by p-hydroxymercuribenzoate indicate the involvement of an --SH enzyme in the carboxylating system.     

Fine needle aspiration cytology of fibrolamellar hepatocellular carcinoma

The major cytologic features seen in fine needle aspirates from two cases of fibrolamellar hepatocellular carcinoma were: liver-like tumor cells, characterized by plump, polygonal forms with eosinophilic, granular cytoplasm; large oval nuclei with extremely prominent solitary nucleoli; and parallel bands of fibrous tissue and fibrocytes seen within the tumor fragments. Other helpful features included intracytoplasmic hyaline globules and well-delineated pale bodies. Clinically, the tumors occurred in young patients with noncirrhotic livers and ran a more favorable course than do other types of hepatocellular carcinoma.     

Effects of G protein and cGMP on phytochrome-mediated amaranthin synthesis in Amaranthus caudatus seedlings

Ｌｉｇｈｔｐｌａｙｓａｃｅｎｔｒａｌｒｏｌｅｉｎｔｈｅｃｏｎｔｒｏｌｏｆｐｌａｎｔｍｏｒｐｈｏｇｅｎｅｔｉｃｒｅｓｐｏｎｓｅｓ.Ｔｈｅｐｈｏｔｏｒｅｃｅｐｔｏｒｓｉｎｃｌｕｄｅｐｈｙｔｏｃｈｒｏｍｅ,ｂｌｕｅｌｉｇｈｔｒｅｃｅｐｔｏｒａｎｄＵＶｒｅｃｅｐｔｏｒｓ,ｏｆｗｈｉｃｈｔｈｅｂｅｓｔｃｈａｒａｃｔｅｒｉｚｅｄｉｓｐｈｙｔｏｃｈｒｏｍｅ.Ａｗｉｄｅｖａｒｉｅｔｙｏｆｍｏｒｐｈｏｇｅｎｅｔｉｃｒｅｓｐｏｎｓｅｓ,ｉｎｃｌｕｄｉｎｇｓｅｅｄｇｅｒｍｉｎａｔｉｏｎ,ｓｅｅｄｉｎｇｄｅｅｔｉｏｌａｔｉｏ…     

Apoplastic calmodulin receptor-like binding proteins in suspension-cultured cells of Arabidopsis thaliana

Calmodulin, a highly conserved protein family that has long been well known as an intracellular calcium sensor, was identified in the culture medium and cell walls of Arabidopsis thaliana suspension-cultured cells by immunoblotting assay. A promotion effect by applying exogenous purified calmodulin and an inhibition effect by the addition of anti-calmodulin anti-serum or calmodulin antagonist to the medium on proliferation of suspension cells were found by monitoring incorporation of [methyl-3H]thymidine into nuclear DNA. Radioligand binding analysis with 35S-labeled calmodulin indicated the presence of specific, reversible, and saturable calmodulin binding sites on the surface of both A. thaliana suspension-cultured cells and its protoplasts; among them at least one is on the surface of Arabidopsis protoplasts, with the Kd approximately 9.2 nM, and two are on the out-surface of Arabidopsis suspension-cultured cells, with Kd values of approximately 47.5 and 830 nM. Chemical crosslinking of 35S-labeled calmodulin to protoplasts revealed 117- and 41-kDa plasma membrane proteins specifically bound to calmodulin, whereas cross-linking with intact suspension-cultured cells verified more calmodulin binding proteins which might be cell wall-associated in addition to membrane-localized. Taking together, our data provide first evidence for the presence of apoplastic calmodulin receptor-like binding proteins on the cell surface of Arabidopsis suspension-cultured cells, which strongly supports our previous idea that apoplastic calmodulin functions as a peptide signal involved in regulation of cell growth and development.